N6-methyl adenosine in siRNA evades immune response without reducing RNAi activity

Abstract

siRNA is a powerful method to suppress specific gene expression and has recently been utilized for molecular biology as well as medicine. However, introduction of dsRNA stimulates immune-responses as side-effects. In the present study, we utilized N⁶-methyl adenosine, one of the natural modified nucleosides, instead of adenosine in siRNA. When adenosine in the passenger or guide strand of siRNA was completely replaced with N⁶-methyl adenosine, the immune response against siRNA was evaded without any reduction in RNAi activity. This knowledge will promote the medical application of siRNA and enhance our understanding on cellular discrimination of non-self and self dsRNA.     

N6-Alkylthiomethyl and N6-Arylthiomethyl Derivatives of Adenosine and Their Cytokinin Activity

Five new derivatives of adenosine, N⁶-[(1-methylethyl)thiomethyl]-(1), N⁶-methyithiomethyl-(2), N⁶-phenylthiomethyl-(3), N⁶-[(3-amino-3-carboxypropyl)thiomethyl]-(4), and N⁶-[(2-amino-2-carboxyethyl)thiomethyl]adenosine (5), were synthesized and their cytokinin activity was tested in the Amaranthus betacyanin assay and the soybean callus growth.

1, 2, and 3 were active in the former assay and all five compounds were active in the latter assay. The activities of the compounds were, however, weaker than those of the reference derivatives, in which Sulfides were replaced by methylenes, N⁶-isopentyl-, N⁶-n-propyl-, N⁶-benzyl-, and N⁶-(5-amino-5-carboxypentyl)adenosine. This fact indicates that the sulfide structure introduced into the N⁶-side chains had the effect of reducing cytokinin activity.     

An Improved Synthesis of N6-(6-Aminohexyl)FAD

Abstract

We report an improved synthesis of N ⁶-(6-aminohexyl)FAD (1) using an efficient one-pot conversion of inosine to the N-trifluoroacetyl protected N ⁶-(6-aminohexyl)adenosine 3. The 5′-O-phosphorylated AMP derivative 4, activated as the imidazolide, was coupled with commercial sodium riboflavin phosphate by using 18-crown-6 in DMF.     

A Functional Screening of Adenosine Analogues at the Adenosine A2B Receptor: A Search for Potent Agonists

Abstract

Various adenosine analogues were tested at the adenosine A_2B receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A_2B receptors. 5′-.N-Substituted carboxamidoadenosines were most potent. 5′-N-Ethylcarboxamidoadenosine (NECA) was most active with an ECso value of 3.1 μM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N⁶) of the purine ring system. The most active N⁶-substituted derivative N⁶-methyl-NECA was 5 fold less potent than NECA. C8-and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3-and 7-deazaanalogues were not active.     

Synthesis of 1,5-Anhydro-2-(N 6-Cyclopentyladenin-9-Yl)-2-Deoxy-D-Altrohexitol

Abstract

The N ⁶-cyclopentyladenosine (CPA) analogue (4) was synthesized in 10 steps starting from glucose. The results of the radioligand binding assays are consistent with the thus far published findings that compounds containing a six-membered moiety at N ⁹ exhibit extremely weak affinity for adenosine receptors. Replacement of the ribofuranosyl moiety of CPA (2) by a 2-deoxy-D-altrohexitol moiety is sufficient to completely abolish its agonist activity.     

Design and Synthesis of A3 Adenosine Receptor Ligands, 2′-Fluoro Analogues of Cl-IB-MECA

Abstract

Synthesis of 2′-deoxy-2′-fluoro-N ⁶-substituted adenosines as bioisosteres of Cl-IB-MECA and their binding affinities to A₃ adenosine receptor are described.     

Design and Synthesis of A3 Adenosine Receptor Ligands, 3′-Fluoro Analogues of Cl-IB-MECA

Abstract

Synthesis of 3′-deoxy-3′-fluoro-N ⁶-substituted adenosines as bioisosteres of Cl-IB-MECA and their binding affinities to A₃ adenosine receptor are described.     

An Efficient Method for the Synthesis of [4–15N]Cytidine, 2′-Deoxy[4–15N]Cytidine, [6–15N]adenosine,and 2′-Deoxy [6–15N]adenosine Derivatives

Abstract

Nucleophilic substitution reactions of 4-azolyl-1 β-P-D-ribofuranosylpyrimidin-2(1H)-one and 6-azolyl-9-β-D-ribofuranosyl-9H-purine derivatives, which were converted from uridine and inosine, with [¹⁵N]phthalimide in the presence of triethylamine or DBU gave N ⁴-phthaloyl[4-¹⁵N]cytidine and N ⁶-phthaloyl[6-¹⁵N]- adenosine derivatives, respectively, in high yields. Similar reactions of those azolyl derivatives with succinimide afforded N ⁴-succinylcytidine and N ⁶-succinyladenosine derivatives in high yields. The corresponding 2′-deoxyribonucleosides were also synthesized efficiently through the same procedure.

     

3′-Amidated 3′-Deoxyxylofuranose Analogues of N 6-−Cyclopentyladenosines: a New Class of Non-Xanthine Antagonists at the Adenosine A1 Receptor.

Abstract

Full adenosine A₁ receptor agonists like CPA and other N ⁶-^?substituted adenosine analogs have previously been shown to become partial agonists upon deletion of the 3′-hydroxyl moiety. The present study further explored the C-3′ site for modification. The modest affinity at A₁ and A_2a receptors found in the 3′-amido-3′-deoxyxyIofuranosyladenine series prompted us to synthesize the corresponding N ⁶-^?cyclopentyl derivatives, which proved to exhibit potent antagonistic behaviour at the A₁ receptors. This represents a new perspective in the purinergic field.     

Synthesis of N 6-Substituted 3′-Ureidoadenosine Derivatives as Highly Potent Agonists at the Mutant A3 Adenosine Receptor

Several N ⁶ -substituted 3 ′-ureidoadenosine derivatives were efficiently synthesized starting from D-glucose for the development of H272E mutant A ₃ adenosine receptor (AR) agonists. Among compounds tested, 3 ′-ureido-N ⁶ -(3-iodobenzyl)adenosine ( 2c ) exhibited the highest binding affinity (K _i = 0.22 μ M) at the H272E mutant A ₃ AR without binding to the natural A ₃ AR.     

Lead Optimization of N 6-Cyclopentyl-3′-amido-3′-deoxyxylofuranosyladenines as Adenosine A1 Receptor Antagonists

Abstract

Strategies toward the further lead optimization of N ⁶-cyclopentyl-3′-amido-3′-deoxyxylofuranosyladenosines as adenosine A₁ receptor antagonists including the synthesis of the 5′-deoxy-analogues and a practical method for parallel amidation are presented.     

Design,Synthesis, and Anti-Tumor Activity of 4′-Thionucleosides as Potent and Selective Agonists at the Human A3 Adenosine Receptor

On the basis of potent and selective binding affinity of Cl-IB-MECA to the human A₃ adenosine receptor, its 4′-thioadenosine derivatives were efficiently synthesized starting from D-gulonic γ -lactone. Among compounds tested, 2-chloro-N ⁶-(3-iodobenzyl)- and 2-chloro-N ⁶-methyl-4′ -thioadenosine-5′ -methyluronamides (7a and 7b) exhibited nanomolar range of binding affinity (K _i = 0.38 nM and 0.28 nM, respectively) at the human A₃AR. These compounds showed anti-growth effects on HL-60 leukemia cell, which resulted from the inhibition of Wnt signaling pathway.     

The Inhibition of Adenosine Kinase by α,ω-Di (Adenosin - N 6- YL) Alkanes1

Abstract

Members of a series of α,ω-di(adenosin- N ⁶-yl)alkanes, comprising two adenosine residues linked with alkyl bridges from 1 to 14 methylene units in length, were found to be inhibitors of rat liver and BHK cell adenosine kinase. The inhibition was competitive with respect to adenosine and non-competitive with respect to ATP. The corresponding α,ω-di(cytidin-N ⁴-yl) alkanes were not inhibitors and N ⁶-alkyladenosines inhibited only weakly.     

N 6-Cyclohexyladenosine Inhibits Veratridine-Stimulated Na Uptake by Rat Brain Synaptosomes

The effect of the stable adenosine analogue, N⁶-cyclohexyladenosine, on ²²Na uptake by rat brain synaptosomes stimulated by veratridine was investigated. In the presence of N⁶-cyclohexyladenosine, both the initial rate and the maximum sodium uptake were decreased. The inhibitory effect of N⁶-cyclohexyladenosine on sodium uptake by synaptosomes after 5 s of incubation with ²²Na was concentration-dependent, antagonized by 1,3-dipropyl-8-p-sulfophenylxanthine, and attenuated by increasing the concentration of veratridine. The possibility that the adenosine analogue, by activating a xanthine-sensitive adenosine receptor, can operate inhibition of the voltage-dependent sodium channels is discussed.     

Deaza-analogues of Adenosine and 2-Chloroadenosine as Agonists of Adenosine Receptors

Abstract

A variety of adenosine analogues have been recently evaluated in order Lo find more potent and selective agonists on adenosine receptors. The most potent adenosine analogues acting on A₁ receptor, a high affinity receptor inhibitory to adenylate cyclase, are N⁶-substituted compounds. So ⁶-cyclohexyladenosine (CHA) and ⁶-L-phenylisopropyladenosine (L-PIA) are extremely potent agonists on A₂ receptor, whereas they are relatively weak agonists on A receptor, a lower affinity receptor which is stirnulatory to cyclase, and they have no effect on the adenosine P site.     

New 2,6,9-trisubstituted adenines as adenosine receptor antagonists: a preliminary SAR profile

A new series of 2,6,9-trisubstituted adenines (5–14) have been prepared and evaluated in radioligand binding studies for their affinity at the human A₁, A_2A and A₃ adenosine receptors and in adenylyl cyclase experiments for their potency at the human A_2B subtype. From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N ⁶ position of 9-propyladenine significantly increased binding affinity at the human A₁ and A₃ adenosine receptors, while the presence of a chlorine atom at the 2 position resulted in a not univocal effect, depending on the receptor subtype and/or on the substituent present in the N ⁶ position. However, in all cases, the presence in the 2 position of a chlorine atom favoured the interaction with the A_2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A_2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles.     

Synthesis and Biological Activity of 6-Hydroxyguanidino-and 6-Hydroxyureidopurine And Their Ribonucleosides

Abstract

N ⁶ ?(1-hydroxyguanidino)purine IIa, and its 9-β-D-ribonucleoside derivative IIb were prepared by reacting at room temperature 6-hydroxyadenine Ia and 6-hydroxyadenosine Ib, with 1-guanyl-3,5-dimethylpyrazole nitrate in DMF. Refluxing IIa and IIb in 95% ethanol gave N⁶?(1-hydroxyureido)purine and its ribonucleoside derivative respectively; the latter compound was also obtained by refluxing Ib with 1-guanyl-3,5-dimethylpyrazole nitrate in ethanol. The two base analogs were inactive against L1210 cells in vitro, but the nucleoside derivatives inhibited the growth of these cells by 50% at 5 × 10 ^-6 and 6 × 10^?7 M respectively. Compound IIb, at 200 mg/kg/day × 5, increased the life span of L1210-bearing DBA/2N mice by 57%. Cytofluorometric determinations showed that IIb inhibited cell growth in the G₂ phase of the cell cycle. also found to inhibit adenosine deaminase activity with a K_i = 3.47 μM.     

Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N ⁶-benzyladenosine, N ⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N ⁶-benzyladenine and 0.01–20 pmol for the N ⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine and their corresponding N ⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N ⁶-benzyladenine, N ⁶-meta-hydroxybenzyl)adenine, and their N ⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N ⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N ⁶-benzyladenosine     

Synthesis of 2′-Deuterio Tubercidin and Adenosine and the Corresponding Arabino Analogs

Abstract

The 2′-deuterio arabino analogs of tubercidin and adenosine have been prepared by Swern oxidation of the 3′,5′-TPDS derivatives of tubercidin and adenosine and reduction with NaBD_4. Subsequent inversion of stereochemistry at C-2′ yielded [2′-²H]tubercidin and [2′-²H]adenosine with 98% deuterium incorporation.     

Radioimmunoassay for N6(methylnitroso)adenosine: production and characterization of antibodies

Antibodies specific for N⁶(methylnitroso)adenosine have been produced in rabbits and a sensitive radioimmunoassay was developed. The nitroso group is immunodominant; 50% inhibition of the binding of [³H]N⁶(methylnitroso)adenosine to antibody was obtained with 9.6 pmoles of N⁶(methylnitroso)adenosine and 200 nmoles of N⁶-methyladenosine. Adenosine was essentially inactive. After nitrosation, N⁶(methylnitroso)adenosine can be detected only in those RNA molecules known to contain N⁶-methyladenosine.